%0 Journal Article
%T Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity
%A John W. Lamppa
%A Margaret E. Ackerman
%A Jennifer I. Lai
%A Thomas C. Scanlon
%A Karl E. Griswold
%J PLOS ONE
%D 2012
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0017042
%X Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017042